The pre-fusion conformation of the RSV F protein (preF) is the primary determinant of neutralization in immunized sera. However, this conformation is unstable and rapidly transitions to the postfusion conformation. A disulfide stabilized prefusion variant with cavity filling mutations (DS-Cav1) has been shown to yield high neutralizing titers in animals; however, it only transiently maintains its native, prefusion conformation. Avatar Medical, LLC has developed a proprietary technology that locks immunogens in native conformations that better present broadly protective epitopes. Locking is accomplished by introducing targeted dityrosine (DT) crosslinks into the fully folded, native protein. We will apply this technology to lock the F protein fully in its pre- fusion conformation (DT-preF) in order to develop a stable, recombinant, preF-based RSV vaccine immunogen. Our minimally modifying, conformational locking technology enzymatically introduces zero-length covalent bonds into proteins and complexes, after the protein has fully folded. Bonds only form between Tyr side-chains in close structural proximity, and thus lock proteins in their native conformation, while preserving structural integrity. DT locking involves 2 steps: (i) expressing and purifying soluble F proteins with targeted, conservative to-Tyr substitutions, and (ii) enzymatically crosslinking the complex in its pre-fusion conformation. Our DT-locked preF immunogens will focus immune responses on potently neutralizing epitopes that are only displayed in the preF conformation, and away from non-or weakly neutralizing, postfusion epitopes. As a result, vaccination with DT-preF should elicit potent and lasting protection against RSV. In this Phase I application, we propose to design and characterize DT-preF variants, and confirm that they maintain the native prefusion conformation, using a panel of preF specific mAbs. We will then demonstrate that our DT-preF immunogen elicits higher neutralizing titers in mice, compared to DS-Cav1. In Phase II we will perform pre- clinical testing in cotton rat challenge studies for efficacy, and in rabbits for safety, with a view toward filing an IND with th FDA. To accomplish the Phase I goals, we will carry out the following Specific Aims: I. Design DT-preF variants in the WT-, disulfide-, and DS-Cav1-stabilized backgrounds, and characterize them antigenically and biochemically by comparison to uncrosslinked and DS-Cav1 controls. (Milestone: Selection of three DT-locked F proteins that retain binding to key preF-specific nAbs.) II. Perform immunogenicity studies in mice, and determine the neutralization titers induced by our DT-preF variants, compared to DS- Cav1, as well as postF, Formalin-inactivated-RSV and live RSV virus controls. (Milestone: Immunization with DT-preF elicits higher neutralization titers than DS-Cav1.)